Early signaling events induced by 280-nm UV irradiation.

The depletion of stratospheric ozone results in increased UV (ultraviolet) light below 300 nm, and has significant effects on biological systems. To better understand the effects of UV in this range, early signaling events induced by monochromatic UV light were investigated using the chicken B cell line DT40 and mutants lacking protein tyrosine kinases (PTKs). Among MAP kinase family proteins, P38 MAP kinase (P38) was selectively and immediately activated by 280 nm UV light in cultured DT40 cells. Activation of P38 was completely inhibited in cells deficient in Lyn and Btk. Introduction of wild-type Btk, but not kinase-inactive Btk, restored the P38 activation. In contrast, P38 activation was not affected in Syk-deficient cells. Tyrosine phosphorylation of Lyn was induced by 280 nm UV light, and pretreatment of cells with orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP), enhanced both Lyn phosphorylation and P38 activation. These results show that Lyn and Btk are upstream regulators of the P38 signaling pathway activated by 280 nm UV light and that the triggering event likely involves inactivation of PTP. Furthermore, cell death induced by 280 nm UV irradiation were augmented by Btk depletion or a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that the Lyn-Btk-P38 pathway promotes cell survival while other Lyn pathways stimulate cell death.     

A catalytic mechanism that explains a low catalytic activity of serine dehydratase like-1 from human cancer cells: crystal structure and site-directed mutagenesis studies

SDH (l-serine dehydratase, EC 4.3.1.17) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes dehydration of l-Ser/Thr to yield pyruvate/ketobutyrate and ammonia. A SDH isoform (cSDH) found in human cancer cell lines has relatively low catalytic activity in comparison with the liver enzyme (hSDH). The crystal structure of cSDH has been determined at 2.8 angstroms resolution. A PLP is covalently attached to K48 by Schiff-base linkage in the active site. The ring nitrogen of PLP is involved in a H-bonding with C309, but is apparently not protonated. Twenty-three amino residues that compose the active site surfaces were identified. The human and rat liver enzymes (hSDH and rSDH) have the same residues, while residues G72, A172, and S228 in cSDH are replaced with A66, S166, and A222, respectively, in hSDH. These residues in hSDH and cSDH were mutated to make complementary pairs of mutated enzymes, and their kinetic parameters were determined. C303 of hSDH and C309 of cSDH which are H-bonding partner of the ring nitrogen of PLP were mutated to alanine and their kinetic parameters were also determined. The crystal structures and the mutation data suggest that having a glycine at residue 72 of cSDH is the major reason for the reduction of catalytic activity of cSDH. Changing alanine to glycine at residue 72 increases the flexibility of the substrate binding-loop (71S(G/A)GN74), so that the bound substrate and PLP are not pushed deep into the active cleft. Consequently, the proton transfer rate from S(G) of C309 to N1 of the bound PLP is decreased, which determines the rate of catalytic reaction.     

Primary structure of myoglobins from 31 species of birds

Primary structure of myoglobins (Mbs) from 31 avian species of 15 orders were reported, although portions of the structures in the 2 species could not be determined. At least 68 of the total 153 amino acid sites were invariant all through the avian, reptilian and human Mbs, and 20 of these sites were "internal", forming the internal hydrophobic cavities in which the heme group remains wrapped. Furthermore, at 27 sites, if replaced, the replacements were mostly conservative, and 13 of the conservative sites were "internal". Thus the all 33 "internal" sites, important for structural and functional stability of the protein, have been well preserved, either invariant or conserved, during evolution from reptiles to birds and mammals. The residue 71 (E14) in 4 penguin species was not deleted as previously reported in emperor penguin Mb but occupied by Gln. The residue 121 (GH3) was deleted in all 3 species studied of Falconiformes. Out of 9 anseriforms, 5 species of different genera showed the identical structure. Secondary structures as viewed by hydropathy profiles were highly similar throughout the reptilian, avian and mammalian Mbs.     

HLA-A*2402-restricted HIV-1-specific cytotoxic T lymphocytes and escape mutation after ART with structured treatment interruptions

Although a limited duration of immune activation of structured treatment interruptions (STIs) has been reported, the immune escape mechanism during STIs remains obscure. We therefore investigated the role of three immunodominant cytotoxic T lymphocyte (epitopes) in 12 HLA-A*2402-positive patients participating longitudinally during the clinical study of early antiretroviral treatment (ART) with five series of structured treatment interruptions (STIs). The frequency of HLA-A*2402-restricted CTLs varied widely and a sustained CTL response was rarely noted. However, a Y-to-F substitution at the second position in an immunodominant CTL epitope Nef138-10 (Nef138-2F), which was previously demonstrated as escape mutation, was frequently detected in seven patients primarily and emerged in the remaining five patients thereafter, and the existence of escape mutations was correlated with high pVL levels early in the clinical course. These findings suggest that escape mutation in the immunodominant CTL epitope may be one of the mechanisms to limit HIV-1-specific immune control in STIs.     

Genome Structure of the Legume, Lotus japonicus

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.Key words: Lotus japonicus, genome structure, Fabaceae, comparative analysis     

Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.     

The association between Japanese primary open-angle glaucoma and normal tension glaucoma patients and the optineurin gene

Glaucoma represents one of the most common eye diseases and is characterized by progressive loss of visual fields. In the more advanced stages bilateral blindness may result, due to optic nerve atrophy and an excavated optic nerve head. Open-angle glaucoma is one of the main disease subsets, which may be further divided into high tension primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG). Recently, the optineurin (OPTN) gene was identified as a causative factor for NTG. Alterations in this gene were found in Caucasian families with NTG. In particular, c.458G>A, c.691-692insAG and c.1944G>A were shown to be risk factors. Since NTG is reported to be the most common form of glaucoma in Japan, and to identify if the OPTN gene plays a role in POAG, the DNAs from 148 unrelated Japanese patients with NTG, 165 patients with POAG and 196 unrelated controls who were not suffering glaucoma were investigated by appropriate genotyping techniques. No glaucoma-specific mutations were found in the OPTN gene in Japanese glaucoma patients. However, some novel single-nucleotide polymorphisms (SNPs) in the exons and introns are reported in this paper for the first time.S. Tang and Y. Toda contributed to the same degree for this study     

Leaf senescence in rice due to magnesium deficiency mediated defect in transpiration rate before sugar accumulation and chlorosis

Magnesium (Mg) is an essential macronutrient supporting various functions, including photosynthesis. However, the specific physiological responses to Mg deficiency remain elusive. In this study, 2‐week‐old rice seedlings (Oryza sativa. cv. Nipponbare) with three expanded leaves (L2–L4) were transferred to Mg‐free nutrient solution for 8 days. In the absence of Mg, on day 8, L5 and L6 were completely developed, while L7 just emerged. We also studied several mineral deficiencies to identify specific responses to Mg deficiency. Each leaf was analyzed in terms of chlorophyll, starch, anthocyanin and carbohydrate metabolites, and only absence of Mg was found to cause irreversible senescence of L5. Resupply of Mg at various time points confirmed that the borderline of L5 death was between days 6 and 7 of Mg deficiency treatment. Decrease in chlorophyll concentration and starch accumulation occurred simultaneously in L5 and L6 blades on day 8. However, nutrient transport drastically decreased in L5 as early as day 6. These data suggest that the predominant response to Mg deficiency is a defect in transpiration flow. Furthermore, changes in myo‐inositol and citrate concentrations were detected only in L5 when transpiration decreased, suggesting that they may constitute new biological markers of Mg deficiency.     

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase is regulated for the accumulation of polysaccharide-linked hydroxycinnamoyl esters in rice (Oryza sativa L.) internode cell walls

Polysaccharide-linked hydroxycinnamoyl esters (PHEs) over-accumulate in the internodes of a rice (Oryza sativa L.) mutant, Fukei 71 (F71). This accumulation is accompanied by over-expression of phenylalanine ammonialyase (PAL). In this study, we show that only one member of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) family expresses in close correlation with PAL. Furthermore, substrate availability to DAHPS is promoted by down-regulating the expression of plastidic pyruvate kinase (PKp), a competitor of DAHPS. Since the over-production of PHEs is caused by D50 gene disruption, these results suggest that specific enzymes in the phenylpropanoid and shikimate pathways are coordinately up-regulated. In addition, the results indicate that carbon-flow into the shikimate pathway is modified for the synthesis of PHEs, and is probably controlled by D50.